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Electrophysiological and Phylogenetic studies on the toxicity of gar oocyte extract Gary LaFleur, Jr Nicole Broussard Chad Loupe Allyse Ferrara Nicholls.

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Presentation on theme: "Electrophysiological and Phylogenetic studies on the toxicity of gar oocyte extract Gary LaFleur, Jr Nicole Broussard Chad Loupe Allyse Ferrara Nicholls."— Presentation transcript:

1 Electrophysiological and Phylogenetic studies on the toxicity of gar oocyte extract Gary LaFleur, Jr Nicole Broussard Chad Loupe Allyse Ferrara Nicholls State University Dept of Biological Sciences Supported by NIH COBRE grant; LSUHSC Walter Ingliss Anderson

2 What makes one fish more persistent than another? Among ancient fishes, gar have maintained a high species count Maisey, 1996 body armor lung egg protection AGWG 2008

3 “There were five persons suffering in all the agony that pain could inflict.” AGWG 2008

4 some ate Garfish Roe paddies were prepared, frozen and fed to crawfish some got mad AGWG 2008

5 Injection of Spotted Gar Roe causes disorientation, rigid paralysis in 60 sec Whole Organism Effect AGWG 2008 We confirmed previoius reports by T Burns BJ Rodrigue Paula Patterson

6 Approach Develop bioassay to test toxicity Determine whether egg envelope is required for toxicity Determine what life stages harbor toxicity Determine species affected by toxicity Determine the mechanism of toxicity Isolate distinct toxic agent Determine whether garfish synthesizes the toxin AGWG 2008

7 Spotted Gar were collected in the Atchafalaya River Basin Photo by C Johanning AGWG 2008

8 Isolation of Ovarian Roe CoBRE 2007 Thanks to Olivia Smith for recent gar ovaries!

9 Making the Ichthyootoxin Extract 2g roe/ 20ml Locke’s Saline Tissue Tearor Homogenize with tubes on ice bath Centrifuged in Sorvall at 10,000 rpm for 10 min Supernatant taken Frozen in -80˚ C Freezer AGWG 2008

10 Saline Control AGWG 2008

11 N=6 N=2N=6 LD 50 approximately 0.6 mg roe / g body wt 1.0 Gar roe (mg / g body wt) 0.20.020.002 Choupique saline 2.0 AGWG 2008

12 Dr. Ferrara using Ovaprim Gar induced to spawn by AGWG 2008

13 Extracts isolated from ovulated eggs, fertilized eggs, hatched embryos all retained toxic activity Photo by R Hotard AGWG 2008

14 -20 0 20 40 60 80 100 120 Injected Substance Percent Survival SalineChoupique Gar roe Unf egg Fert egg 5-day larva N=2N=6 2.0 1.0 mg / g body wt N=2 All stages tested contained toxicity Chorions not necessary for effect AGWG 2008

15 Uca longisignalis Uca panacea Procambarus clarkii Toxin Sensitive Toxin Insensitive Poecillia latipinna Fundulus grandis Paralysis Results AGWG 2008 Allyse suggested: “why not use a crustacean with a clear carapace?”

16 Assaying Effect on Neurogenic Heart 18 sec AGWG 2008 Nicole Broussard MS student

17 Crawfish Preps for Electrophysiology AGWG 2008 Thanks Hamilton Farris, LSUHSC neuron, tail muscle, and heart muscle preps tested

18 Extracellular recordings suggest a disruption of glutamate signaling at neuromuscular junctions AGWG 2008

19 Sensitive Groups: Vertebrates (except teleost) CRUSTACEANS Tested the toxin sensitivity of other invertebrates, within and without Phylum Arthropoda We expected that the toxin would affect both the mollusk and the insect MODEL SPECIES: Littorina irrorata Marsh Periwinkle (Snail) Gryllus sigillatus Tropical house cricket Gilbert 2000 SNAILS … ? CRUSTACEANS: YES INSECTS … ? VERTEBRATES: YES EXCEPT FOR TELEOST Phylogenetic tests

20 Average Snail Survival Data compiled from two trials consisting of 30 animals each. Hourly observations noted at 4, 8, and 20 hours post-injection Error bars denote standard deviation. Littorarina irrorata Positive Control in Crawfish = 10 mg / g body wt Five snails/group n= 2 Chad Loupe BS 2007

21 Average Cricket Survival Compiled data from four cricket trials comprising of 30 animals each. Hourly observations noted at 4, 8, and 20 hours post-injection Error bars denote standard deviation. Gryllus sigillatus Positive Control in Crawfish = 10 mg / g body wt Five Crickets/Group N=4

22 After six trials and over 120 total observation hours, the cricket and snail had no notable response to the toxin Toxin had a profound effect on crustaceans. Immediate paralysis and eventual death.. Although it is known that glutamate is used as a neurotransmitter in insects and snails, the toxin had no effect. Although it is known that glutamate is used as a neurotransmitter in insects, the toxin had no effect on crickets. crustaceans may be the only invertebrates affected SNAILS? CRUSTACEANS: YES INSECTS: NO VERTEBRATES: YES EXCEPT FOR TELEOST

23 Conclusions Paralysis and Cardiotoxicity Assay developed All crustaceans tested are sensitive The toxin does not affect any teleosts tested Egg Envelopes not necessary, cytoplasmic localization Electrophysiology prep suggests neuromuscular junction Neither Snails, nor Insects sensitive to toxin Unanswered Neurotoxic mechanism of action Specific molecular entity Biosynthetic origin of extract AGWG 2008

24 Research: Applications similar to other neurotoxins for experimental manipulation Crustacean avoidance: The species selectivity of the garfish toxin may provide anti-fouling agents against barnacles. Ecology: concentrating the toxin in eggs may have contributed to the extraordinary persistence of this ancient fish group. Possible Research Applications AGWG 2008

25 Questions? Walter Ingliss Anderson Thanks Allyse and Quenton for a great meeting!

26

27 EXPERIMENT 2: Isolating the Toxic Constituent of the Extract Hypothesis: The neurotoxic entity contained within the garfish roe homogenate is a single compound that can be isolated Design: Homogenate will be fractionated and each fraction tested. The toxic fraction will be further separated by SDS gel electrophoresis isolating single protein bands for further voltage clamp testing. Rationale: If the neurotoxin is indeed heat-labile as previously shown, then we should be able to identify it using protein electrophoresis. Other toxins such as tetrodotoxin and ciguateratoxin are NOT proteins, and so there is a remote possibility that this compound will be a molecule other than a protein, wherein isolations may depend on GC, Mass Spec or other chromatography methods Future Experiments

28 EXPERIMENT 3: Elucidating the site of Synthesis of the Neurotoxin Hypothesis: We hypothesize that the garfish is synthesizing this proteinaceous neurotoxin, and secreting into the blood, where it is subsequently concentrated in the roe. Design: PCR primers and antibodies will be designed against the N- terminal sequence, allowing for RT-PCR cloning of the complete cDNA coding for the protein. Rationale: The site of synthesis can be shown by documenting RNA levels in specific tissues. Additionally immunolabeling cytoplasmic vesicles containing the protein would suggest synthesis though it may also suggest storage, as in the ovary. If no tissues in the garfish are found to contain RNA coding for the neurotoxin, a microbial origin of the neurotoxin will be implicated. Future Experiments

29 EXPERIMENT 1: Which ion channel is perturbed by roe homogenate? Hypothesis: The paralysis caused by garfish roe injection in crawfish is due to the specific blocking of an ion channel Design: Using a crawfish whole cell prep that Dr. Farris' lab has already had experience with, we will test the whole homogenate, and then separate fractions of the garfish roe extract Rationale: If paralysis is caused by blocking of an ion channel we will be able to record this and identify the channel using voltage clamp techniques. If the crawfish prep is not convenient, other crustacea will suffice, as well as cell preps from tetrapods or cultured cells Future Experiments using electrophys AGWG 2008

30 Our plan for the next several months includes: (1) Collaborate with Dr. Farris at LSUHSC to document the activity on isolated crustacean and molluscan neurons. (2) Test whether garfish toxin affects other invertebrates including insects and molluscs. (3) Fractionate oocyte extract and test in paralytic, cardiotoxic and neurophysiological bioassays. (4) Obtain N-terminal and internal sequence (5) Design degenerate primers to isolate the cDNA. AGWG 2008

31 EXPERIMENT 4: Obtaining the Primary Sequence of the Neurotoxin Hypothesis: By obtaining the N-terminal sequence of the neurotoxin we will have information needed to design primers for further cloning and sequencing of the cDNA, as well as information that may suggest whether the molecule originates from the garfish or a symbiotic microbe. Design: Proteins are routinely N-terminally sequenced, often receiving up to 20 amino acids If the N-terminus of the protein is blocked, proteolytic cleavage products can be submitted as well for “internal sequences” Future Experiments

32 Future Work Fractionation followed by bioassays to identify the active molecule N-terminal sequencing to test whether conserved with other agent neurotoxins Determine if toxin originates from garfish or symbiont Behavioral studies of crawfish to test whether it deters predation Walter Ingliss Anderson

33 Paralysis, and death also oocurs in U. panacea, and U. longisignalis WAS ’07 Ancient Fishes

34 1851 Report of human poisoning Crawfish paralyzed by roe injection B. Background and Significance Research: Applications similar to Tetrodotoxin for channel manipulation Patents: The species selectivity of the garfish toxin may provide anti- fouling agents against barnacles. Evolution: Garfish currently being considered for protection intriguing reproductive strategy may deter predation.

35 Injection, tachycardia, arrest in 10 sec Real time visualization of extract Injection into shrimp cephalothorax AGWG 2008

36 Symbiotic microbe? 1. How does roe cause paralysis? 2. What is 1 o structure of toxin? 3. Is toxin synthesized by garfish? N C Current Research Plan AGWG 2008


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